Optical detection and analysis of particles

ABSTRACT

Method and apparatus for the single particle detection of submicron structures such as biological molecules and viruses utilises an optical element ( 100 ) comprising an optically transparent substrate ( 1 ) partially coated with a thin film of metal ( 2 ) illuminated with an optical beam ( 4 ) incident on a non-metal coated region ( 3 ) of the surface of the optical element at a point adjacent or close to the metal coated region of the optical element such that the beam propagates above but close and substantially parallel to the metal surface defining a measurement zone from within which submicron particles ( 7 ) contained in a sample ( 6 ) placed in contact with the optical element scatter or emit light which can be detected in the far field by conventional photodetection systems. The apparatus can be configured in a flow cell or optical microscope configuration.

This application is a continuation of U.S. patent application Ser. No.10/1513,160, filed Oct. 29, 2004, now U.S. Pat. No.7,399,600, which isthe national filing of International Application No. PCT/GB03/01827,filed Apr. 29, 2003, claiming priority to British Application No.0209666.7 filed Apr. 29, 2002.

The present invention relates to the optical detection and analysis ofparticulates of nanometer, sub-micron or micron dimensions.

A large number of principles and techniques exist by which particles canbe analysed in terms of their number, size, shape, composition andmotion. Historically, the observation and characterisation of particleslies in the domain of microscopy in which highly magnified images ofparticles are generated through the use of high powered lensed systemsand which can be seen directly by eye or can be captured by camera forsubsequent interpretation by the operator or by an image analysissystem.

There are many types of microscope systems capable of characterising theparticle in terms of its interaction with the incident illumination. Forinstance, the particle may selectively absorb certain wavelengths of thelight such as in differential absorption, the technique most common inconventional transmission microscopy. Other microscopical variants existwhich selectively monitor specific wavelengths generated by the particlewhen illuminated by the incident illumination, such as fluorescentmicroscopy which is useful in reducing background interference and whichcan be used to identify specific structures through the use offluorescent labels. Yet other microscopical techniques utilise the wayin which the particle induces a phase shift in the incident light, suchas phase contrast or interference microscopy. Other microscopetechniques, such as epiluminescent microscopy, employ light scatteringat high angles to allow low contrast particles to be visualised againstnon-illuminated background. Other similar versions of this technique areused in microscopy, of which the most common is referred to as darkfield microscopy. In this case, the sample is illuminated by a highnumerical aperture source and the central portion of the illuminatingcone is blocked from entering the detection objective by an optical stopso that the particle is illuminated at an oblique angle only.

Methods of illumination vary greatly and in certain circumstances thesample (typically an aqueous suspension of particles) can be placed on atransparent (typically glass or silica) optical substrate which isilluminated by a suitably defined and collimated optical beam at acertain angle called the critical angle at which the incident light isrefracted along the plane of the optical element on which the liquidsample is placed. A small portion of the beam, called the evanescentwave, propagates a small distance into the sample phase above theoptical substrate and particles entering this evanescent region act toscatter some of this otherwise non-radiative field. The light coupledout (i.e. scattered by the particle within the evanescent field) canthen be detected in the far field either by eye or by a suitabledetector situated normal or at high angle to the plane of the surface.When employed in a microscope configuration this technique is referredto as evanescent field microscopy and relies on the principle offrustrated total internal reflection

Numerous non-imaging methods exist for the optical analysis ofsuspensions μm or sub-μm particles or solutions of nm scale particlessuch as biological molecules or macromolecules. Many such techniquesmonitor the interaction between biological molecules and in order todefine a region within such interactions can be specifically detectedwithin minimal interference from other species in the bulk of thesolution phase, such analyses are frequently carried out at theinterface of an optical waveguide or fibre optic structure onto thesurface of which have been immobilised biological capture molecules suchas antibodies, specific for the target analyte. In conventionalwaveguide or fibre optic systems use is made of the changes in therefractive index properties at the surface interface following bindingof specific biological molecules in the surface associated evanescentfield region of the optical structure. This field extends, however, onlysome 100-200 nm into the bulk solution phase and is accordingly limitedin its ability to monitor weak interactions involving limited numbers ofmolecular interactions. Such a method is disclosed in DE 4307042(930305), in which a single or multilayer of receptor molecules aredeposited on an evanescent waveguide sensor device and which is capableof sensing and quantifying various chemical and biochemical species insolution. A similar method is disclosed in WO 9005295 claiming priorityof SE 884075 (881110) in which a wedge shaped prism is used to allowlight reflected at different angles off the underside of the opticalsensor element to be imaged and analysed to quantify specific species insolution. Similarly, EP677735 claiming priority of U.S. Pat. Nos.228,233 (940,415) describes an optical resonator cavity in which lightis reflected from a total internal reflector (TIR) cavity in contactwith a solution components of which interact with the evanescent fieldwithin the TIR cavity allowing quantification of species in thesolution. These techniques are characterised by their reliance on theanalysis of light which is reflected from the underside of a sensingelement surface.

The ability to follow such low numbers of interactions or binding eventscan, however, be significantly enhanced, by one or two orders ofmagnitude, by employing Surface Plasmon Resonance techniques in whichthe surface of the optical waveguide structure is coated with a thinfilm of a conductive metal, typically gold, silver or aluminium, inwhich electromagnetic waves, called Surface Plasmons, can be induced bya beam of light incident on the metal-glass interface at a specificangle called the Surface Plasmon Resonance angle. Modulation of therefractive index of the interfacial region between the solution and themetal surface following binding of the captured macromolecules causes achange in the SPR angle which can either be measured directly or whichcauses the amount of light reflected from the underside of the metalsurface to change. Such changes can be directly related to the mass andother optical properties of the molecules binding to the SPR devicesurface. Several biosensor systems based on such principles have beendisclosed. Thus WO 9005305 claiming priority of SE884074 (881110)describes the use of a metal film deposited on one side of a block unitof optical instrumentation, one multi-functionalised side of which is incontact with a solution of reagents or samples to be measured, the otherside is illuminated by an optical beam within the block unit of opticalinstrumentation caused to reflect off the metal surface at an angle suchthat reflectance is modified by selective binding of ligands on thefunctionalised surface. Measurement of the reflected beam can becorrelated to concentrations of specific species binding to thefunctionalised sensor surface.

Similarly EP 341927 claiming priority of GB881154 (880510) describes abiological or biochemical testing sensor comprising a surface plasmonresonance (SPR) sensor and a sample-antibody surface arranged toinfluence resonance characteristics. The SPR sensor comprises ametallised glass slide onto the glass-metal interface of which isdirected a beam of light at an angle at which surface plasmons areinduced to resonate in the metal film. Changes in the resonance angle onbinding of analyte are determined by measuring the intensity or angle ofthe light internally reflected from the metal-glass interface. Suchnon-imaging reflectance techniques monitor only the binding ofrelatively large numbers of macromolecules through measurement ofchanges in the amount or position of the reflected light

As with the evanescent techniques described above, these techniques arecharacterised by their reliance on measuring the intensity of lightreflected from the surface or changes in the resonance angle on bindingof specific sample components.

A modification of such SPR devices has been described which can be usedto locate, visualise, detect or count the presence of individualmacromolecules or very sub-μm particulates in which the optical effectof interaction of individual m or very sub-μm scale structures with theevanescent surface plasmon resonance field causes light to be scatteredfrom such structures into the far field at high angles. Thus WO 98/57148and U.S. Ser. No. 09/308,049 claiming priority of PCT/GB98/01591describes a method and apparatus for the single particle detection ofsubmicron structures such as biological molecules and viruses whichutilises an optically transparent substrate coated with a thin film ofmetal which is illuminated with an optical beam incident at surfaceplasmon resonance angle wherein submicron particles contained in asample placed in contact with the metal film scatter light which can bedetected in the far field by conventional photodetection systems. Theapparatus can be configured in a flow cytometric or optical microscopeconfiguration.

Similarly, WO 98/22808 and WO0142768 describe a surface plasmonresonance apparatus for detecting a soluble analyte (e.g. a protein) ora particulate analyte (e.g. a cell), the apparatus comprising a sensorblock adapted to receive a sensor, said sensor, for example a sensorslide, having a metallized sensor surface capable of binding theanalyte; a light source capable of generating an evanescent surfaceplasmon resonance wave at the sensor surface of a sensor slide on thesensor block; a first detector capable of detecting light from the lightsource which is internally reflected from the, sensor surface; and asecond detector (e.g. a video camera) capable of detecting lightscattered or emitted from an analyte bound thereto. Optionally theapparatus further comprises a second light source for increasing theintensity of the light scattered or emitted from an analyte bound to thesensor surface, preferably, this is sited to such as to minimize theamount of light transmitted therefrom which is detected by the firstdetector. Also disclosed are sensors adapted for use in the apparatus,and methods of detecting analytes in samples comprising exposing samplesto the sensor surface of the apparatus.

In both the above cases employing the surface plasmon resonancephenomenon, it is necessary for the illuminating beam to be incident onthe underside of the metal film in contact with the underlying opticallytransparent substrate in order to induce surface plasmons at the metalsurface, energy from the evanescent field associated with which is thenscattered into the far field by the target particulates within theevanescent field. Furthermore, the angle at which the incidentillumination must strike the underside of the metal film in order toexcite surface plasmons in the metal film surface is very narrow andrequires careful alignment of the component optical elements. Finally,the amount of energy coupled through the metal from the incidentilluminating beam to the evanescent surface plasmon field is frequentlyinsufficient for the particles interacting with said field to scattersufficient energy to the detectors to allow very small, for example lessthan 200 nm, particles to be visualised. Thus WO0142768 is limited todescribing the visualisation of bacterial cells by this technique by wayof example, such particles being in excess of 500 nm in diameter.

If generation of images of individual particles is not required, forinstance when it is necessary only to determine the presence orotherwise of particles and/or to estimate their size, size distribution,number etc., then other principles in which light scattering phenomenapredominate may be used. Such methods rely on the measurement of theamplitude of optical signal generated by the interaction of particleswith suitably intense and focused beams of light (typically from lasersources), each particle passing through the optical measurement zone inwhich the interrogating beam is caused to pass, signals generated by theinteraction of the particle with the optical beam being detected bysuitable photosensitive devices such as photomultiplier tubes,photodiodes, CCDs and the like.

Such instruments are referred to as particle detectors or particlecounters and are used widely in a variety of industrial and scientificapplications. One such technique, known as flow cytometry, allowsparticles in a concentrated suspension, to be addressed on an individualbasis by diluting the sample through adding it slowly to a rapidlyflowing hydrodynamic sheath of substantially particle free liquid, theoutput of which is directed by a finely adjusted nozzle to flowaccurately through a finely focused interrogating laser beam whichconstitutes a measurement volume. By measuring the intensity ofscattered light and where applicable, the fluorescence wavelengthgenerated by the interaction of the particle with a suitably focused andintense optical source, particles as small as 0.2 μm can be quantifiedand various optical parameters relating to their size and differentialabsorption or fluorescence characteristics can be determined.

Below a certain particle size limit, however, the signal generated bythe interaction of the particle with the interrogating beam of light isinsufficient for it to be distinguished from the background inherent insuch optical light scattering instrument configurations. Increasing theintensity of the interrogating optical beam acts merely to increase theintensity of background as well as the signal generated by the particle.To determine the presence of such very sub-micron particles it isnormally usual to employ higher resolution non-optical techniques suchas electron microscopy but these suffer from significantly highercomplexity and cost.

There is therefore a need for a simple, robust and low cost opticalparticle detection system capable of detecting the presence of verysmall particles (such as those, for example, substantially below ¼ ofthe wavelength of illuminating radiation) on an individual basis withoutthe need for expensive, high powered and hazardous optical sources,which does not rely on the phenomenon of scattering of the evanescentfield associated with resonant surface plasmons. Preferably suchapparatus should be compatible with existing optical microscopes andparticle detection apparatus, simple to use and operate and capable offurnishing information, such as particle size, size distribution, numberand other optical parameters on suspensions of particles of mixedcharacteristics in a frequently complex background.

For the avoidance of doubt, the content of all publications mentioned inthis specification is incorporated herein by reference.

SUMMARY OF THE INVENTION

The present invention is based at least in part on the unexpectedfinding that when a small volume of a suspension of sub-micronparticulates, exemplified by virus particles, is placed onto the surfaceof an optical element comprising, for example, an optically transparent(typically glass or silica) substrate, one part of the surface of whichhas been coated with a thin (e.g. 10's nm) film of metal, for examplechrome, silver or gold which is at least partially optically opaque,such that a further adjacent region of the optically transparentsubstrate is left uncoated by the metal film and which non-metal coatedregion is illuminated by a beam of light caused to be incident on it, orin close proximity to it, at a point which is close (e.g. within 5 mm,preferably within 1 mm, more preferably within 0.5 mm) to (but typicallynot coincident with) the metal film coated region and at an angle suchthat the optical beam is caused to propagate, by refraction, through thesample substantially parallel to, and at a small distance above, theadjacent metal film, individual sub-micron particles within the path ofthe beam passing through the sample above the metal film are found toscatter sufficient amounts of light so as to be individually discerniblethrough a conventional microscope objective/lens combination, by eye orby a suitable photodetector such as a photon multiplier tube, solidstate photodiode, CCD camera or other photosensitive device placed in animage plane in the far field and normal or at high angle to the plane ofthe otherwise non-radiative metallised surface. The particles capable ofbeing thus individually detected are of a dimension such that they wouldnot otherwise have been detectable by conventional optical transmission,dark-field, phase contrast, evanescent field or surface plasmonresonance microscopical techniques such as those described above.

The sample will typically, but not necessarily, comprise a liquidcomprising a suspension or other dispersion of particles. The particlesmay be solid or may conceivably be liquid (e.g. fine droplets in anemulsion). A liquid sample will normally, but not inevitably, be anaqueous liquid.

In general terms, the present invention provides apparatus for thedetection and/or analysis of individual sub-micron particles in asample, the apparatus comprising: a substrate, means for illuminatingthe substrate with a focused optical beam, and means for individuallydetecting the optical radiation from individual particles in a sampleplaced on the coated portion of the substrate surface through theirinteraction with the optical beam.

Components generally suitable for use in the apparatus of the inventionare disclosed in, inter alia, U.S. Pat. No. 6,280,960.

The person skilled in the art will understand that, whilst preferablythe substrate per se is wholly or substantially transparent to the beamof electromagnetic radiation, the opaque coating is sufficient to rendersubstantially or wholly opaque that portion of the substrate the surfaceof which is so coated.

Typically the beam of electromagnetic radiation is caused to be incidentupon the substrate surface opposed to that which is partially coatedwith the opaque coating, the beam then being propagated along thedesired path, through the sample, by refraction through the substrate,it therefore being a requirement in such an embodiment that thesubstrate is wholly or substantially transparent to the electromagneticradiation, and that at least a portion of the surface of the substrateis not covered with the opaque coating.

It is at least conceivable however that in other embodiments the beam ofelectromagnetic radiation may be directly incident upon the samplewithout having first passed through the substrate e.g. by passing thebeam in close proximity to the substrate directly parallel to the coatedsurface of the substrate or at a slight “grazing” angle thereto, or inanother embodiment that the electromagnetic radiation is caused to passthrough the sample by reflection from the upper surface of the substrate(i.e. that the beam is incident upon the same side of the substrate asthat which is at least partially coated with the opaque coating). Inthese embodiments there is no requirement that the substrate be whollyor partially transparent and no requirement that at least a portion ofthe substrate surface is left uncoated.

The electromagnetic radiation will typically have a wavelength in therange 1×10⁻⁸ to 1×10⁻⁵ nm (i.e. in the range from far ultra-violet todeep infra-red). Conveniently the electromagnetic radiation will belight of a wavelength visible to the human eye (i.e. in the range fromabout 3 to 8×10⁻⁷ nm). The electromagnetic radiation will convenientlybe a focused beam of visible light, and the apparatus willadvantageously comprise suitable lens or other focusing means. Asuitable electromagnetic radiation source is a laser.

Typically the substrate will comprise glass or silica and be opticallytransparent at wavelengths visible to the human eye.

In some embodiments it is preferred if the opaque coating is reflective.The inventor has so far found an opaque metallic coating to beparticularly suitable. The thickness of the opaque coating is preferablyless than 500 nm, more preferably less than 250 nm, and most preferablyless than 100 nm.

The edge of the beam of electromagnetic radiation, defined by its 1/e²point, is preferably caused to propagate less than 2 μm above the coatedsurface of the substrate, more preferably less than 1 μm and mostpreferably less than 500 nm. Further, the beam is preferably caused tobe propagated substantially parallel to the coated surface of substratei.e. at an angle to the plane of the surface of less than 5.degree.,preferably less than 2.degree., more preferably less than 1.degree., andmost preferably less than 0.5.degree., and the term “slight angle”should be construed accordingly for the purposes of the presentspecification.

The invention also provides a corresponding method.

In a preferred embodiment according to the present invention there isprovided a method and apparatus for the individual optical detectionand/or characterisation of small (relative to the wavelength of lightbeing used for illumination) particles suspended in a transparent liquidor gas medium (e.g. for the purposes of determining particlecharacteristics such as size, size distribution, number concentration,shape or other optical characteristics such as fluorescence,polarisation, phase modulating properties, etc.) wherein a samplecontaining the suspended particles is placed onto the surface of anoptical element comprising an optically transparent (e.g. glass orsilica) substrate, one part of the surface of which has been coated witha thin (10's nm) film of metal, for example chrome, silver or gold whichis at least partially optically opaque, such that a further adjacentregion of the surface of the optically transparent substrate is leftuncoated by the metal film and which non-metal coated surface region isilluminated by a beam of light caused to be incident on it at a pointwhich is close to (but typically not coincident with) the adjacent metalfilm coating and at an angle such that the optical beam is caused topropagate, by refraction, through the sample substantially parallelwith, or at a slight angle to, the plane of the metal film and at asmall distance above the metal film, such that individual sub-micronparticles within path of the optical beam passing through the sampleabove the metal film are found to scatter sufficient amounts of light soas to be individually discernible through a conventional microscopeobjective/lens combination, by eye or by a suitable photodetector suchas a photon multiplier tube, solid state photodiode, CCD camera or otherphotosensitive device placed in an image plane in the far field andnormal or at high angle to the plane of the coated surface. Theparticles capable of being thus individually detected are of a dimensionsuch that they would not otherwise have been detectable by conventionaloptical transmission, dark-field, phase contrast, evanescent field orsurface plasmon resonance microscopical techniques.

Advantageously, the presence of the opaque film below the path that isdescribed by the optical beam acts to enhance the visibility of theparticles resident therein, such particles, when below a certaindimension, scattering insufficient light to be visible when the metalfilm is absent. Accordingly the presence of an optically opaque (orsubstantially opaque) film on at least part of the surface of thesubstrate appears to be an essential feature of the invention.

Fluorescence emitted by, or light scattered from, the particles is seen,by eye or by suitable detectors, as a point of light arising from eachparticle in the measurement region, the amplitude of signal from each ofwhich can be indicative of various optical properties of the particlesas well as indicating its presence, size, motion, number, concentration,fluorescence, etc. all parameters of which can be quantified, ifdesirable, by suitable signal processing or image analysisinstrumentation.

In accordance with the invention, microscope optics and instrumentationcan be used to allow particles so small as to be otherwise undetectableby conventional optical microscopy techniques to be individuallydetected for the purposes of determination of particle presence, size,particle size distribution, concentration, number, fluorescentattributes (whether inherent or through the addition of fluorescentlabels) for measurement of specific parameters associated with theparticle composition, polarisation modifying properties, phasemodulating properties or any other parameter normally addressable byoptical methods of analysis.

In the particular case where the present method and associated apparatuscould be used in conjunction with a non-microscopical application, suchas in the case of a particle counting apparatus for the purposes of e.g.determining particle count as a function of size class, the particlesuspension can be caused to flow over the surface of the optical elementsuch that particulate suspensions that would otherwise be so dilute asto contain too few particles within the measurement volume forstatistically accurate estimation of particle presence or numberconcentration or size distribution to be made, an increased volume ofthe particle-containing medium can be caused to flow over the detectionregion thereby increasing the number of particles capable of beingaccurately detected and analysed. For particle counting and analysis onsuch larger volumes the present method may be employed in an opticalparticle measurement and analysis system exemplified by thoseinstruments known as flow cytometers in which a suspension of particlesis caused to pass through an optical measurement region by introducingvia a nozzle, the particulate-bearing sample into a stream ofsubstantially particle free fluid moving at a higher velocity, known asa hydrodynamic sheath, such that the particle-bearing sample is dilutedto the point where particles pass through the optical measurement regionon an individual basis and the direction of the flow of particle-bearingsample can be finely adjusted to be optimally aligned with the opticalmeasurement region. One type of flow cytometer employs what is known asa jet-on-an-open-surface (JOOS) configuration in which the samplebearing hydrodynamic sheath flow is played onto a flat, opticallytransparent surface such that the position of the sample stream can befinely adjusted through adjustment of the nozzle position and flowvelocity to be more accurately placed at the waist of the interrogatingoptical beam. In accordance with the invention the use of a metallisedoptical surface, as described herein, advantageously allows smallerparticles than would otherwise be optically detectable to be visualisedin such a system by virtue of the enhancement of visibility afforded bythe presence of the metal film as described above.

The process of the invention may thus be used to determine particlepresence, size, particle size distribution, concentration, number,fluorescent attributes (whether inherent or through the addition offluorescent labels) for measurement of specific parameters associatedwith the particle composition, polarisation modifying properties, phasemodulating properties or any other parameter normally addressable byoptical methods of analysis but which is particularly useful forcarrying out such analyses on particles that are so small as to beotherwise undetectable on an individual basis by optical systemsincorporating bulk lens configurations such as conventional microscopes,flow cytometers or other optical particle measurement instruments.

In this regard it has been found that the present invention allowssub-micron particles such as unlabelled viruses in solution orsuspension to be directly visualised and counted on an individual basisthrough the use of optical sources of moderate power such as solid statelaser devices of mW output.

The present invention, by virtue of its sensitivity to detection ofparticle-associated events close to a surface, further allows theinteraction of sub-micron particles with surface coatings andfunctionalised layers to be individually monitored and analysed in time.Such events may include the interaction of discrete virus particles witha coating on the optical element specifically designed to substantiallyreproduce the properties exhibited by a cell surface for the purposes ofinvestigating virus-cell envelope infection events.

Similarly, in accordance with the invention the adhesion of sub-micronregions of cell membranes or walls and regions thereof with surfaces,chemically or biochemically modified or otherwise, may be monitored atresolutions and sensitivities exceeding those afforded by conventionaloptical microscopic techniques. Advantageously, such events can bemonitored in real time and in an aqueous environment unlike thoselyophilised conditions necessary for visualisation of such interactionsby electron microscopy.

The range of the types of particle which can be individually seen by theprocess of the invention is also varied and broad. The use of theoptical element herein described, by virtue of its ability to facilitatethe generation of detectable optical signals from sub-micronparticulates, allows the process of the invention to be applied to theestimation of contaminant levels in process or industrial fluids andliquids which are desired to be contaminant free, the detection of virusparticles and other sub-micron biological entities in biological,environmental, biotechnological, foodstuff and clinical samples, such asblood and urine and other body fluids, purification media,pharmaceutical preparations, foodstuffs and the like. Very sub-micronparticles designed to act as fluorescent labels, such as those referredto as quantum dots, are equally amenable to detection and analysis.Other particulates in solution or suspended in a fluid phase that may beindividually detected, counted and characterised in accordance with theinvention include contaminating organic or inorganic particles inotherwise particle free fluids, smoke or other combustion productparticles in gases, contaminants in oils, micro-emulsion (oil in wateror water in oil) droplets, liposomes and vesicles, micelles,sub-microscopic cells such as mycoplasmas, colloids of natural orindustrial origin, or any suspension, colloidal fluid or preparation inwhich light scattering centres exist and which are too small too beanalysed by conventional optical instrumentation. It will, of course, beappreciated that the process of the invention allows any particulatecapable of scattering or modifying radiation incident upon it and whichcan be distinguished from the background by a suitable detector to beindividually detected and analysed.

It will be further appreciated that the process of the invention isapplicable to the analysis of individual macromolecules andmacromolecular constructs which, through labelling with a suitableoptical amplifier or fluorescent label capable allowing them to bedistinguished from the background if required, would not otherwise bedetectable on an individual basis using conventional optically basedparticle characterising instrumentation. Equally the invention may beapplicable in situations wherein the detectable particles are part of alarger supramolecular structure, such as a cell or cellular component, abiofilm, polymeric layer or the like.

The use of the partially metallised optical element illuminated with asuitable optical source as described herein is particularly advantageousin that readily available light sources of modest power such as low costgas, diode or solid state lasers can be used in conjunction withconventional detection optics and electronic photosensitive devices todetect particles which normally would only be capable of beingindividually visualised by very much more sophisticated and complextechniques such as electron microscopy.

It should be noted that the invention is not confined to the case wheresamples are in solution. Where they are however, the solvent need not bewater or even liquid but the solution may take the form of any formknown to physical chemistry in which particles can be opticallydifferentiated from their surrounding environment for analyticalpurposes. Furthermore, it should be clear that the process of theinvention can be applied to situations in which the particulates to bedetected and individually visualised are acted on by other physicalforces, such as electrical or acoustic fields, so as to, for instance,induce physical motion or separation from other constituents in thesample.

Besides the embodiments of the method and the apparatus described abovethe invention can be used in a variety of other configurations and for avariety of other purposes. Thus, besides the incorporation of theinvention into a JOOS type flow cytometric configuration it could beincorporated into any other optical detection apparatus in which theinteraction of very small particles with an optical field is measured.For instance, the invention could be incorporated into a scanning probemicroscope, such as a scanning near-field microscope, as a means ofvisualising a surface and locating desirable or interesting features onthat surface to assist in the efficient high resolution scanning andimaging of the surface by the scanning probe tip.

Similarly, the invention could be used to monitor and analyse thedynamic Brownian motion of the particles thus visualised, informationfrom which can, by suitable analytical techniques such as numberfluctuation spectroscopy and fluorescence correlation spectroscopy orpoint tracking image analysis equipment, be used to derive a range ofparticle characteristics such as size and size distribution, number,concentration and the nature and dynamics of particle-particleinteractions or interactions of the particles with a functionalisedlayer, if present.

Similarly, the invention could be used to enhance the performance of andderive more information from other analytical techniques such as SurfacePlasmon Resonance (SPR) apparatus allowing SPR device surfaces to besimultaneously analysed for particulates in any given sample underanalysis.

It should also be noted that small particulates interacting with theoptical field present at or close to the surface of the optical elementdescribed herein, can be subject to physical motive forces from thelight itself, a phenomenon known as photophoresis. This ability tomodify the physical motion of particles, for instance effectively trapthem in a certain location by the pressure of light alone, could be usedto advantage in the analysis and manipulation of particles in accordancewith the invention.

Particular benefits which ensue from the invention include the abilityto directly and individually visualise sub-microscopic particles such asviruses and other particles in the 5-500 nm diameter range which havenot necessarily had to be optically amplified by use of fluorophore orlight scattering labels and which would not otherwise be detectable byconventional microscope instrumentation. Analytical resolution isgreatly improved by the ability afforded by the invention tocharacterise and analyse on a particle-by-particle basis a population ofparticles that may be diverse in size and optical properties and, ifdesired, to determine the spatial distribution of said particles.Furthermore, the ability to monitor the dynamic behaviour of particlessuspended in a liquid by sophisticated analytical techniques such asdigital or optical correlation of signals emanating from the particlesis valuable in determining other physical properties and characteristicsof submicron particles that might not otherwise be obtainable by othertechniques. The physical components from which the apparatus can beassembled are not complex or expensive and can be used by non-expertusers. The apparatus lends itself to being retrospectively fitted to arange of existing optical analytical instrumentation and instrumentationdesigns to improve resolution and performance.

The invention will now be described in more detail by way of examplewith particular reference to the accompanying schematic drawings ofwhich;

FIG. 1 illustrates apparatus according to the invention for thedetection of sub-micron particles such as viruses suspended in anaqueous fluid.

FIG. 2 illustrates one use of the invention for application in a flowcytometric configuration.

FIG. 3 illustrates the use of the apparatus for studying the interactionof particles with a functional layer deposited on the optical element.

EXAMPLES

Referring to FIG. 1, apparatus in accordance with the inventioncomprises an instrument element 100 having an optically transparentsubstrate 1, typically a glass or silica prism or flat, onto part ofwhich is deposited a thin film of metal 2, typically 30-80 nm depth ofe.g. gold, silver, aluminium or chrome deposited by any suitablesputtering, vapour phase, electrochemical or other deposition means. Theoptical substrate is only partially covered by the metal film 2, aportion 3 of the surface being left uncoated. A light beam of suitablecollimation, intensity, polarisation and wavelength or wavelength range4 is focused by lens 5 to be incident on the optical element such thatthe beam strikes the surface of the optical element in the region 3which is not covered by the metal fil 2 but which is adjacent to themetallised region at an angle at which, when a sample of liquid 6containing a suspension of particles 7 is placed onto the surface of theoptical element 100, the beam is caused, by refraction, to propagatethrough the sample substantially parallel to and a small distance abovethe metal film. Those particles 7, present within the beam individuallyact to scatter light which can be detected in the far field by asuitably aligned and focused lens arrangement 8 such as a microscopeobjective, which could be an immersion lens, and associated lenses to besubsequently observed by eye or analysed using a photosensitive deviceand suitable signal processing or image analysis instrumentation.Alternatively, the particles could be viewed through a suitableoptically transparent planar window in, for instance, a flow cellarrangement or through a microscope cover slip or equivalent.

It will of course be understood that besides the simple observation oflight scattered by particles 7, other optical consequences of theirbeing illuminated by the beam 4 when propagating close to the metal film2 may be observed and analysed. Thus, if the particle population 7 iscomprised entirely or partially of particles which are inherentlyfluorescent or have been specifically labelled through the use ofselected fluorescent labels, those particles which fluoresce on cominginto close proximity to the region of the metal coated substrate 2illuminated by beam 4, may be specifically observed through the lensassembly 8 if the image is first filtered by a suitable fluorescencefilter assembly 9.

It will be further understood that the use of several differentfluorescence filters will allow multiple wavelengths to be separatelyanalysed extending the information that can be obtained about a multiplystained particle suspension under view.

In FIG. 2 is shown an alternative apparatus for use in a flow cytometricconfiguration of the jet-on-an-open-surface type. The optical elementshown in FIG. 1 can be mounted in such a way as to allow a stream ofparticle bearing sample to be flowed across its surface. When ahydrodynamically focused stream of fluid 13 (a “hydrodynamic sheath”)emanating from a nozzle 14 and containing a stream of particles 15introduced into the hydrodynamic sheath fluid by tube 16 is passedthrough the region at which the optical beam propagates over themetallised region of the optical element 100, particles directed, byfine adjustment of nozzle 14, to flow in close proximity to this regionwill either scatter light or be induced to fluoresce, the opticalradiation of which is detected, through use of lens system 17 containinga fluorescent filter assembly 18, if required, by a suitablephotosensitive detector and associated signal processing electronicscapable of measuring the optical signal generated by the separateparticles at a rate which allows particles to be analysed individuallyand sequentially at high rates, typically hundreds or thousands persecond. The lens assembly 17 can be designed and constructed such thatone or more of a variety of angles of scattered light can be selectedfrom the scattered radiation or fluorescence emanating from theparticle.

In FIG. 3 is shown an alternative configuration for the detection ofinteraction of particles with a functional surface comprising aninstrument element 100 as shown in FIG. 1 coated with a functional layer30 which may comprise a polymeric or biological material whichsubstantially replicates the properties exhibited by a natural cellmembrane or wall surface and from which the interaction of particles 31in a liquid sample 32, which may, for instance, be infective virusparticles, can be obtained information about the rate, number andbehaviour of binding events between the particles and the functionalsurface. Alternatively, the functional layer may comprise a chemicallyor biochemically modified layer onto which have been attached chemicalor biological molecular moieties such as antibodies or other selectiveligand binding structures which exhibit a specific affinity for targetmolecular or particulate structures 31 the presence and number or otherproperty of which is required to be established in the sample 32.

As in the other embodiments described above, it is of course obviousthat besides the simple observation of changes in light scattered byparticles 31, other optical consequences of their coming into closeproximity to the region of the metal/glass interface illuminated by theoptical beam may be observed and analysed. Thus, if the particlepopulation 31 is comprised entirely or partially of particles which areinherently fluorescent or have been specifically labelled through theuse of selected fluorescent labels, those particles which fluoresce oncoming into close proximity to the region of the metal/glass interfaceilluminated by the optical beam may be specifically observed through thelens and detection assembly if the image is first filtered by a suitablefluorescence filter. Similarly, rotation of the polarisation of theincident beam by the particles can be measured in this invention.

It will be further understood that the use of several differentfluorescence filters will allow multiple wavelengths to be separatelyanalysed extending the information that can be obtained about a multiplystained particle suspension under view.

In the preferred embodiment, the optical element is a silica quartzplanar substrate onto which has been deposited by a sputtering method,an approximately 50-80 nm thick layer of chrome. The optical element isilluminated at suitable incident angle by a laser beam of modest power,for example 40 mW and suitable wavelength, for instance 488 nm. A dropof a biological sample such as a sample of clinical or biological origindiluted in phosphate buffered saline containing a population ofunlabelled refractile virus particles of clinical or biotechnologicalsignificance (such as adenoviruses) is placed on the optical elementsurface and the light the virus particles scatter as they move underBrownian motion within the optical beam propagating through the samplein close proximity to the metallised region of optical element isobserved by eye down a conventional microscope fitted with a ×40immersion objective. Images of the virus can, of course, be captured onfilm or on video recording by suitable instrumentation for subsequentviewing and analysis. The presence and number concentration of virusparticles in the sample can be determined from the intensity of lightthey scatter (light scattering in this size region being a strongfunction, for instance radius, of their size) or from counting thenumber of points of light of intensity associated with a particle sizeper unit volume in the sample for any given illumination intensity.

It should be understood that though this invention has been described byway of various examples a variety of modifications are possible withoutdeparting from the scope of the appended claims.

1. Apparatus for the optical detection and/or analysis of individualsub-micron particles, the apparatus comprising: a substrate, part of onesurface of which is coated with a film comprising an optically opaquemetal, and part of which surface is left uncoated, such that the surfacehas a coated portion and an uncoated portion; means for illuminatingsaid substrate with a focussed beam of radiation from a source, suchthat a focussed optical beam is caused to be incident on the substrateat a point on the uncoated portion of the surface adjacent to, but notcoincident with, the metal film coated portion of the surface, such thatthe optical beam is caused to propagate above but substantially parallelor at an angle of less than 5° and close to the surface of the metalfilm; and means for individually detecting, by a lens and detectorarrangement situated in the far field at the normal or high angle to theplane of the metal film, the optical radiation individually scatteredby, or otherwise caused to emanate from, individual particles in asample placed on the coated portion of the substrate surface throughtheir interaction with the optical beam.
 2. Apparatus according to claim1, wherein the source is a laser.
 3. Apparatus according to claim 1,wherein the substrate is part of a larger assembly designed andconstructed for purposes other than particle detection.
 4. Apparatusaccording to claim 1, wherein the lens and detector arrangementcomprises a conventional microscope.
 5. Apparatus according to claim 1,in combination with a flow cytometer instrument.
 6. Apparatus accordingto claim 1, wherein the substrate comprises or is formed from a whollyor partially optically transparent material.
 7. Apparatus according toclaim 1, wherein the focussed optical beam is incident upon one surfaceof the substrate, is refracted during passage through the substrate, andemerges at the opposite surface at a slight angle adjacent or close tothe metal film coated portion of said surface.
 8. A microscope or flowcytometer comprising apparatus in accordance with claim
 1. 9. Apparatusaccording to claim 1, wherein the focused optical beam is caused to beincident on the substrate at a point on the uncoated portion of thesurface within 5 mm of the metal film coated portion of the surface.